U.S. patent number 4,797,259 [Application Number 06/941,386] was granted by the patent office on 1989-01-10 for well-type diagnostic plate device.
This patent grant is currently assigned to Pall Corporation. Invention is credited to Peter J. Degen, Jerold Martin, Vlado I. Matkovich.
United States Patent |
4,797,259 |
Matkovich , et al. |
January 10, 1989 |
Well-type diagnostic plate device
Abstract
A diagnostic test device is provided including a plate having at
least one well, preferably a plurality of wells, each with an open
bottom across which a composite membrane comprising three layers is
placed, with a hydrophobic, liquid-tight seal provided at the
periphery of each well. The composite membrane from the top of
upstream side to the bottom or downstream side, in sequence,
includes a first reaction or filtration layer formed from a thin,
liquophilic, microporous membrane, a second or sealing layer,
preferably a hydrophobic material in sheet or fiber form, such as
nonwoven polypropylene fibers, and a liquophobic, preferably
hydrophobic, barrier layer having one or more apertures which
allows liquid to exit the well while eliminating lateral migration
of a pendant liquid drop. The liquophobic seal provided by the
liquophobic sealing layer eliminates "cross-talk" by lateral
diffusion or wicking.
Inventors: |
Matkovich; Vlado I. (Glen Cove,
NY), Martin; Jerold (New York, NY), Degen; Peter J.
(Huntington, NY) |
Assignee: |
Pall Corporation (Glen Cove,
NY)
|
Family
ID: |
25476389 |
Appl.
No.: |
06/941,386 |
Filed: |
December 15, 1986 |
Current U.S.
Class: |
422/534; 356/246;
422/552; 422/948; 435/288.3; 435/7.92 |
Current CPC
Class: |
B01D
61/18 (20130101); B01L 3/50255 (20130101); G01N
33/54366 (20130101) |
Current International
Class: |
B01D
61/18 (20060101); B01L 3/00 (20060101); G01N
33/543 (20060101); C12M 001/20 () |
Field of
Search: |
;422/101,102 ;435/7,301
;356/246 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
0098534 |
|
Jan 1984 |
|
EP |
|
86/07606 |
|
Dec 1986 |
|
WO |
|
Primary Examiner: Richman; Barry S.
Assistant Examiner: Johnston; Jill
Attorney, Agent or Firm: Leydig, Voit & Mayer
Claims
We claim:
1. A diagnostic test device comprising:
a plate,
at least one well formed in said plate, said at least one well
having an open bottom, and
a composite membrane having at least three layers including,
sequentially, a microporous liquophilic reaction layer, a sealing
layer, and a liquophobic barrier layer having at least one aperture
therein for each said well and located substantially at the center
of the well, said composite membrane coextensive with the bottom of
said at least one well and forming a liquophobic, liquid-tight seal
at the periphery of the well.
2. The diagnostic test device of claim 1 wherein said reaction
layer comprises a hydrophilic material.
3. The diagnostic test device of claim 1 wherein said sealing layer
comprises a synthetic, thermoplastic web of microfibers.
4. The diagnostic test device of claim 1 wherein said liquophobic,
liquid-tight seal is a hydrophobic liquid-tight seal.
5. The diagnostic test device of claim 1 wherein said reaction
layer includes a hole located at substantially the center of said
well.
6. The diagnostic test device of claim 1 wherein said microporous
reaction layer comprises a biologically inert polymeric
material.
7. The diagnostic test device of claim 1 wherein said microporous
reaction layer is formed from a polyamide or polyvinylidene
difluoride.
8. The diagnostic test device of claim 1 further including a test
reagent immobilized on said reaction layer.
9. The diagnostic test device of claim 1 wherein said barrier layer
is formed from a material having a greater liquophobicity than that
of either the reaction layer or the sealing layer.
10. The diagnostic test device of claim 1 wherein said at least one
aperture consists of one aperture.
11. The diagnostic test device of claim 1 wherein said three layers
are in intimate contact with one another.
12. The diagnostic test device of claim 11 wherein said three
layers are bonded to one another.
13. The diagnostic test device of claim 1 wherein said at least one
well comprises a plurality of wells.
14. The diagnostic test device of claim 13 wherein said composite
membrane covers and is substantially coextensive with the bottoms
of all of said wells.
15. The diagnostic test device of claim 1 wherein said device is
adapted to be operatively associated with a differential
pressure.
16. The diagnostic test device of claim 15 wherein said
differential pressure is a reduced pressure.
17. The diagnostic test device of claim 1 wherein said microporous
reaction layer comprises a hydrophilic, surface-modified
polyamide.
18. The diagnostic test device of claim 17 wherein said
surface-modified polyamide is a surface-modified nylon 66.
19. The diagnostic test device of claim 1 wherein said sealing
layer comprises a hydrophobic material.
20. The diagnostic test device of claim 19 wherein said hydrophibic
sealing layer is formed from a polyamide, a linear polyester, a
polyolefin, a copolymer of two or more olefins, or mixtures of the
aforementioned polymers.
21. The diagnostic test device of claim 20 wherein said hydrophobic
sealing layer comprises polypropylene.
22. A diagnostic test system comprising:
(1) a diagnostic test device including:
(a) a plate,
(b) at least one well formed in said plate, said at least one well
having an open bottom, and
(c) a composite membrane having at least three layers including,
sequentially, a microporous liquophilic reaction layer, a sealing
layer, and a liquophobic barrier layer having at least one aperture
therein for each said well and located substantially at the center
of the well, said composite membrane coextensive with the bottom of
said at least one well and forming a hydrophobic, liquid-tight seal
at the periphery of the well; and
(2) means for generating a differential pressure operatively
associated with said diagnostic test device.
23. The diagnostic test system of claim 22 wherein said at least
one aperture consists of one aperture.
24. The diagnostic test system of claim 22 wherein said
differential pressure generating means comprises a vacuum manifold
adapted to be operatively associated with a means for generating a
reduced pressure.
25. The diagnostic test system of claim 24 wherein said vacuum
manifold is in air-tight sealing relationship with said diagnostic
test device.
26. The diagnostic test system of claim 24 wherein said vacuum
manifold is formed integral with said diagnostic test device.
27. A diagnostic test device comprising:
(a) a first plate,
at least one well formed in said first plate, said at least one
well having an open bottom, and
a composite membrane having at least three layers including,
sequentially, a microporous, liquophilic membrane reaction layer, a
sealing layer, and a liquophobic barrier layer having at least one
aperture therein for each said well and located substantially at
the center of the well, said composite membrane coextensive with
the bottom of said at least one well of said first plate and
forming a liquophobic liquid-tight seal at the periphery of the
well; and
(b) a second plate in liquid receiving relationship with said first
plate,
at least one well having an impermeable bottom in said second plate
to receive liquid from said at least one well in said first
plate.
28. The diagnostic test device of claim 27 wherein said at least
one aperture consists of one aperture.
29. A diagnostic test system comprising:
(1) a diagnostic test device including:
(a) a first plate,
(b) at least one well formed in said first plate, said at least one
well having an open bottom, and
(c) a composite membrane having at least three layers including,
sequentially, a microporous, liquophilic membrane reaction layer, a
sealing layer, and a liquophobic barrier layer having at least one
aperture therein for each said well and located substantially at
the center of the well, said composite membrane coextensive with
the bottom of said at least one well of said first plate and
forming a liquophobic liquid-tight seal at the periphery of the
well;
(2) means for generating a differential pressure operatively
associated with said diagnostic test device; and
(3) a second plate located intermediate and in operative
relationship with said diagnostic test device and said differential
pressure generating means, said second plate in liquid receiving
relationship with said first plate,
at least one well having an impermeable bottom located in said
second plate to receive liquid from said at least one well in said
first plate.
30. The diagnostic test system of claim 29 wherein said at least
one aperture consists of one aperture.
31. The diagnostic test system of claim 29 wherein said
differential pressure generating means comprises a vacuum manifold
adapted to be operatively associated with a means for generating a
reduced pressure.
32. The diagnostic test system of claim 31 wherein said vacuum
manifold is in air-tight sealing relationship with said diagnostic
test device.
33. The diagnostic test system of claim 31 wherein said second
plate is located within said vacuum manifold.
34. The diagnostic test system of claim 31 wherein said vacuum
manifold is formed integral with said diagnostic test device.
35. The diagnostic test system of claim 34 wherein said second
plate is located within said vacuum manifold.
Description
TECHNICAL FIELD
The present invention relates to a diagnostic device useful in
chemical and particularly biological and biochemical assays. The
invention is particularly directed to multiple well filtration
devices, such as microtitration plates, able to retain fluids for
extended periods of time and, under specified conditions, to remove
liquid quickly and completely.
BACKGROUND ART
Diagnostic devices, including test plates, and, in particular,
multiple well or microtitration plates, have been used for both
quantitative and, especially, qualitative chemical and biological
tests for decades. Various designs and configurations have
proliferated as the area of enzyme immunoassays has expanded. Test
devices and, particularly, plates having a plurality of wells which
include microporous membrane filters, have also become routinely
used in clinicallaboratories in recent times. This has resulted, at
least in part, from development of cell and tissue culture
techniques and assays in fields such as virology and
immunology.
It is common in the clinical assay to simultaneously run a number
of different tests on the same liquid sample, to run duplicate
tests, or to perform the same test procedure on a number of
different samples. In such instances, it is preferred to employ a
multiple well filtration plate, such as a ninety-six well plate.
Such test devices have advantages in that they provide a single
test apparatus rather than multiple test apparatus and also provide
side-by-side comparison of test results within a single device.
Such plates, however, have several significant shortcomings. Many
of the materials used to form at least the bottom portion of such
test devices are porous in nature and permit liquid in the wells to
pass through the bottom either by gravity flow or capillary action.
Although such liquid loss may be permissible and even desirable in
many instances, uncontrolled loss of fluid in many assays leads to
inaccurate or unreliable results. This is particularly true in
treating or conducting tests on living cells or tissues. In such
applications, the biological material is frequently grown or
maintained in media of specified composition for periods of from
several hours to several days. Losses of even small volumes of
liquid can in some instances alter the results drastically.
A second common problem encountered with the use of such multiple
well test plates involves a phenomenon known by some as
"cross-talk". Such occurrence involves the migration of liquid,
sometimes in the form of a pendant drop suspended from the bottom
of one well, to an adjacent well. Two causes of the type of
migration known as cross-talk are (1) wicking of fluid or diffusion
of solutes laterally through the membrane between adjacent wells,
and (2) coalescing of pendant drops suspended below the wells. Such
migration may lead to spurious results, both when the liquid
removed from the wells is to be analyzed or, possibly, when liquid
flows back into an adjacent well.
DISCLOSURE OF THE INVENTION
The present invention is directed to a diagnostic test device which
includes a plate having at least one well, and preferably a
plurality of wells, each well having an open bottom. At the bottom
of the well and forming a hydrophobic, liquid-tight seal at the
periphery thereof is placed a composite membrane comprising three
layers which are, preferably, in intimate contact with one another.
Proceeding from the top or upstream side to the bottom or
downstream side of the composite membrane, in sequence, the first
layer is a reaction or filtration layer formed from a thin,
liquophilic microporous membrane, such as a membrane of filtration
material. After transfer of a test sample to a well and removal of
liquid, in those situations when subsequent reactions are performed
on substances retained by the composite membrane, it is the
reaction layer which generally forms the site at which reaction
occurs. Placed below the reaction layer is a second or sealing
layer. This second, preferably porous layer functions as both a
means of securing or adhering the reaction layer to a liquophobic
barrier layer as well as forming a liquophobic seal at the
periphery of the well where the side walls of the well contact the
composite membrane. Because of the liquophobic seal, cross-talk by
lateral diffusion or wicking is eliminated. Preferred is a
hydrophobic material for the second layer. The third or downstream
layer is a liquophobic barrier layer. This layer includes a small
aperture located substantially at the center of the well. This
barrier layer substantially eliminates dripping, enhances isolation
of each well in multiple well devices, and inhibits lateral
migration of a pendant liquid drop from one well to another
well.
In addition to the aforementioned advantages, the multiple well
diagnostic plate device of the present invention permits greater or
enhanced sensitivity in tests performed with the device as a result
of the increased surface area afforded by the porous nature of the
reaction layer. In some instances, the substantially increased
sensitivity resulting from the substantially higher surface area of
the microporous membrane provides results making the difference
between operability and inoperability of the particular protocol.
Furthermore, the rapid and efficient removal of sample and reagent
solutions only after application of a pressure differential permits
the solutions to be removed rapidly and completely without the use
of a pipette.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates a preferred embodiment of the present invention
in combination with a manifold.
FIG. 2 is a sectional view of FIG. 1 taken along line II--II.
FIG. 3 is a sectional view of an embodiment of the present
invention which includes an upper well-containing plate portion
having a composite membrane at the bottom opening of each well and
a lower receptacle portion.
BEST MODE FOR CARRYING OUT THE INVENTION
The Diagnostic Plate
The diagnostic plate employed in the present application may have a
variety of forms and be made from any suitable material. Preferred
materials are thermoplastic resins including polyolefins, such as
polypropylene, and polystyrene. The plate may contain as few as one
well or as many wells as can be arranged within the plate and
effectively serviced by the source of differential pressure, i.e.,
reduced or positive pressure, employed. Filtration plates currently
being used typically have ninety-six wells and this number of wells
is not incompatible with the present invention. Typically, the
wells in such devices are closely spaced for efficiency and
economics. As a result, the potential for cross-talk in such
devices is enhanced.
The wells themselves may be formed in the shape of cylinders
projecting upwardly from a base of the plate or, as preferred, may
be formed as cylinders projecting downwardly from an upper surface
or plane of the plate. Each of the wells may have vertical or
downwardly tapered side walls, the latter forming conical or
tapered wells.
The dimensions and shape of each well may be varied depending upon
the application of the device. Thus, the well will typically have a
cylindrical configuration but other configurations, such as a
square or rectangle, may also be used. Likewise, the diameter of
the well may vary considerably depending upon such factors as the
size of the sample to be used in each well and whether the device
is to be used for diagnostic purposes, its most widespread
anticipated application, or for separation and isolation of
substances.
The size of the liquid sample used and, therefore, the dimensions
of the well may also depend on the sensitivity of the reaction
performed in the well when the device is used for diagnostic
purposes. Typically, the diameter of a well will be from about 1 mm
to about 100 mm. Preferably, the diameter of a well will be about 2
mm to about 25 mm. Typically, the height of a well is about 1 to
about 75 mm, preferably about 5 to about 10 mm.
The bottom of each well has an opening formed therein. For example,
the well may have the configuration of an open cylinder or of a
hollow truncated or frustoconical cone. The periphery or brim of
the opening to which the composite membrane is adhered generally
forms a horizontal surface.
Composite Membrane
Across the bottom of the well and sealed in liquid-tight
relationship to the periphery of the well is a composite membrane.
The composite membrane comprises at least three layers. The first
or uppermost layer constitutes a "reaction layer" which, when the
device of the present invention is used for diagnostic purposes, is
the layer in or upon which reagents are added and tests are
performed. This layer may also be used as a filtration medium to
separate liquid and solid components when the material retained by
the composite membrane is not intended for further
characterization. The material from which this layer is formed,
like the other layers, must not react adversely with substances
found in either the samples, reagents or solvents employed in the
analyses. In addition, the reaction layer must be formed from a
liquophilic, microporous membrane, typically having an absolute
pore rating of about 0.001 to about 20 microns, preferably about
0.02 to about 8 microns, and most preferaly about 0.2 to about 3
microns. The reaction layer preferably is also skinless. Materials
which are suitable for use as the reaction layer also have voids
volumes in the range of about 60 to about 90 percent, preferably in
the range of about 75 to about 90 percent. Preferred materials are
hydrophilic in nature and are, therefore, easily water-wettable and
tend to freely pass aqueous solutions. Examples of liquophilic
materials which may be used in the present invention include, but
are not limited to, polyamides, such as nylon 66, polyvinylidene
difluoride, cellulose esters, and nitrocellulose.
Liquophilicity, as used herein, refers to the wettability of the
membrane by the liquid(s) with it is contacted. The wettability or
liquophilicity of a solid structure, e.g., a membrane, is a
function of that structure's critical surface energy and the
surface tension of the applied liquid. If the critical surface
energy is at least as high as the surface tension of the liquid,
the liquid will spontaneously wet the solid structure. For example,
a microporous membrane having a critical surface energy of 72
dynes/cm or higher will be wetted by water which has a surface
tension of 72 dynes/cm, i.e., it is hydrophilic.
The capability of a porous structure (membrane) to be wetted by a
liquid can be determined by placing a drop of liquid on the porous
structure. The angle of contact provides a quantitative measure of
wetting. A very high angle of contact indicates poor wetting, while
a zero angle of contact defines complete or perfect wetting.
Materials used in the subject invention as the wettable or
liquophilic porous layer are characterized by being readily or
spontaneously wetted by the applied liquid and have a low angle of
contact with the applied liquid. Indeed, when a drop of a test
liquid(s) is placed on a spontaneously wettable or liquophilic
microporous membrane layer, the drop of liquid penetrates the layer
amd wets the membrane, effectively providing a zero angle of
contact therewith.
Suitable materials should also be capable of being treated with and
retaining or immobilizing a substance being analyzed and/or a
reactant which may be used to perform a specified test or reaction
with the substance being analyzed for in a sample. The reactant,
which may be of ionic, molecular, or macromolecular nature may be
immobilized on the reaction layer by strong physical forces or by
being bonded in some manner, such as covalent chemical coupling, to
the surface of the microporous, liquophilic reaction membrane
layer. As employed herein, the term "surface" or "surface area"
refers not only to the gross surface(s) of the structure but also,
in those cases where a microporous structure such as a membrane is
under consideration, to the surfaces of the micropores, i.e., the
interior surfaces of the structure which are contacted by fluid
during use. As distinguished from "surface area" or "surface", the
exposed planar or gross area of the material is herein referred to
as the "reaction layer area", "microporous reaction layer area",
"reaction layer membrane area", or the like.
Wettability or liquophilicity is a requisite of the materials used
for the microporous reaction layer of the present invention. It is
particularly preferred that such materials be capable of being
spontaneously wetted. Some of the materials which are suitable or
preferred for use as the reaction layer in the present invention
are intrinsically hydrophilic or water-wettable. Others may be
modified to render them hydrophilic. BIODYNE.RTM. is an N66
polymide, microporous membrane commercially available from Pall
Corporation which is inherently water-wettable by virtue of its
method of manufacture (see U.S. Pat. No. 4,340,479).
Polyvinylidene fluoride membranes are not inherently water-wettable
but can be rendered such by an appropriate surface treatment.
Microporous, polyvinylidene fluoride membranes which have been
treated to render them hydrophilic are commercially available. As
discussed above, wettability or liquophilicity is a function of the
critical surface energy of the solid structure and the surface
tension of the liquid. Wettability may also be expressed in terms
of intrusion pressure which may be defined as the applied pressure
required for liquid to penetrate into the pores of the membrane.
Although a function of the properties of the liquid used, such as
surface tension, materials which are particularly preferred for the
reaction layer of the composite membrane have intrusion pressures
of or close to zero.
Materials which are preferred for the reaction layer also have
large surface areas. This feature permits a greater amount or
higher concentration of reactant to be immobilized in the reaction
layer. Accordingly, higher sensitivities may be achieved using the
test plate of the present invention.
Polyamides preferred for use in the present invention include
nylons of the type described in
U.S. Pat. No. 4,340,479, which is incorporated herein by reference.
As noted above, a membrane material of this description which is
particularly useful for the present invention is a microporous,
hydrophilic nylon membrane commerically available from Pall
Corporation under the trademark BIODYNE.RTM..
Another preferred membrane useful as the reaction layer is
IMMUNODYNETM.TM., available from Pall Corporation. IMMUNODYNETM.TM.
is a modified CARBOXYDYNE.RTM. membrane, also available from Pall
Corporation. CARBOXYDYNE.RTM. is a hydrophilic, microporous,
skinless nylon 66 membrane with controlled surface properties
formed by the cocasting process described in U.S. Pat. No.
4,707,266, as discussed below, specifically by cocasting nylon 66
and a polymer containing an abundance of carboxyl groups to form a
membrane having controlled surface properties characterized by
carboxyl functional groups at its surfaces. IMMUNODYNETM.TM.
membranes may be prepared from CARBOXYDYNE.RTM. membranes by
treating them with trichloro-s-triazine in the manner described in
U.S. Pat. No. 4,693,985, discussed below.
Also included among the preferred polyamide membranes for the
present invention are hydrophilic, microporous, skinless polyamide
membranes with controlled surface properties of the type described
in (1) U.S. patent application Ser. No. 850,061, filed Apr. 7,
1986, now U.S. Pat. No. 4,707,266, which is a continuation
application of U.S. patent application Ser. No. 459,956, filed Jan.
21, 1983, now abandoned, which in turn is a continuation-in-part
application of U.S. patent application Ser. No. 346,118, filed Feb.
5, 1982, now abandoned and in (2) U.S. patent application Ser. No.
848,911, filed Apr. 7, 1986, now U.S. Pat. No. 4,702,840, which is
a continuation application of U.S. patent application Ser. No.
460,019, filed Jan. 21, 1983, now abandoned which is a
continuation-in-part application of U.S. patent application Ser.
No. 346,119, filed Feb. 5, 1982, now abandoned.
All of the aforementioned U.S. patent applications are specifically
incorporated herein by reference. These hydrophilic, microporous,
substantially alcohol-insoluble polyamide membranes with controlled
surface properties are formed by cocasting an alcohol-insoluble
polyamide resin with a water-soluble, membrane-surface-modifying
polymer having functional polar groups. Like the preferred
hydrophilic, microporous nylon membranes which do not have
controlled surface-modified polar groups present, the polyamide
membranes of the present invention having controlled surface
properties are also skinless; that is, they are characterized by
through pores extending from surface-to-surface which are of
substantially uniform size and shape. If desired, however,
materials having tapere through pores, i.e., pores which are larger
at one surface of the sheet, narrowing as they approach the
opposite surface of the sheet, may be used.
The surface-modifying polymers used to prepare the polyamide
membranes with controlled surface properties, useful in the present
invention, comprise polymers which contain substantial proportions
of chemically functional groups, such as hydroxyl, carboxyl, amine,
and imine groups. As a result, the membranes include, at their
surfaces, high concentrations of functional groups such as
hydroxyl, carboxyl, imine, or a combination of any of the above
groups which do not react with one another. These polyamide
membranes having controlled surface properties have higher
concentrations of carboxyl or imine groups at their surfaces than
the preferred microporous, hydrophilic, skinless polyamide
membranes described above which do not have controlled surface
properties, i.e., those which are formed from the preferred
polyamide resin but are not cocast with surface-modifying
polymer.
The reaction layer may be treated by any method known to one of
skill in the art to deposit and/or bind reagents thereto. As
indicated above, the reagent may be of an ionic, molecular, or
macromolecular nature. When used as a diagnostic tool to provide a
visible change, the reagent may be one or a combination of
substances which is initially colorless and which, upon reaction
with a suitable material, provides an optically measurable
response. Other possible variations include the use of suitable
labels, such as the formation between the deposited reagent and the
material for which testing is being conducted of a complex or
compound which is appropriately labeled by any known technique,
such as enzymatic/substrate labels or the like.
Although treatment of the reaction layer with a suitable reagent(s)
may be performed at the time at which diagnostic tests are to be
performed, including addition of the test reagent(s) both
immediately preceding and following introduction of the sample
containing the analyte to the well(s), the present invention is
expected to have greatest applicaion to, and a preferred embodiment
includes, a composite membrane in which the reaction layer has been
pretreated with at least one test reagent. Typically, pretreatment
is conducted after the composite membrane has been sealed to the
wells but before the device is shipped to a user. If the reagent(s)
is not heat sensitive, the membrane may be treated before
assembling the composite membrane.
A useful method of binding reagents of a molecular nature,
especially macromolecules, and particularly those of a biological
nature, is disclosed in U.S. Pat. No. 4,693,985, specifically
incorporated herein by reference. This patent describes a method
for immobilizing a wide range of biologically active substances as
acceptor molecules on active membranes. The acceptor-bound
membranes described in the application are capable of immobilizing
and binding a wide variety of biologically-active compounds,
specifically ligands, to the acceptor molecules. Using such
reaction layers or membranes permits the testing of bodily fluids,
such as blood, serum, plasma, urine, saliva, and the like, and
testing for particular substances by chemical assays or
immunoassays, such as those where a specific label is employed,
such as one indicating enzyme activity or an electromagnetic energy
absorbing and/or emitting label, such as a fluoroescent label. The
macromolecules used as reagents and bound to the reaction layer or
which are assayed for using the device of the present invention
generally include materials of a biological nature and are
frequently proteinaceous in nature. The reagent or acceptor
molecule bound directly to the reaction layer or the ligand being
tested for include such substances as immunoglobulins or
antibodies, either polyclonal or monoclonal, antigenic substances,
apoproteins, receptors, glycoproteins, lectins, carbohydrates,
hormones, enzymes, carrier proteins, heparin, coagulation factors,
enzyme substrates, inhibitors, cofactors, nucleic acids,
etcetera.
Placed below, preferably in intimate contact with or, most
preferably, adhered, bonded, or otherwise secured to the reaction
layer, particularly at the periphery of each well, is a porous
sealing layer which serves several purposes. Strongly preferred for
use as a sealing layer is a very porous liquophobic structure. The
term "liquophobic" as used herein is effectively the obverse of the
term "liquophilic", that is, a liquophobic material has a critical
surface energy lower than the surface tension of the applied liquid
and is not readily or spontaneously wetted by the applied
liquid(s). Liquophobic materials are characterized, then, by a high
contact angle between a drop of liquid placed on the surface and
the surface. Such a high contact angle indicates poor wetting. The
porous sealing layer assists in bonding the reaction layer to the
barrier layer. It also makes it possible to form a liquophobic seal
at the periphery of the well, thereby eliminating cross-talk by
diffusion or wicking. The use of such material in the sealing layer
also provides the composite membrane with the ability to withstand
or maintain a pressure differential. For example, it prevents,
without the application of vacuum or high pressures, penetration of
liquid through the composite membrane and the concomitant loss of
liquid from the well by dripping. Thus, at specified pressure
conditions, generally close to or at atmospheric pressure, liquid
is retained within a well. However, when reduced pressure is
applied to the downstream side of the composite membrane or super
atmospheric pressure is applied to the upstream side of the
membrane, liquid readily drains from the well.
Materials suitable for use as the sealing layer, in addition to
being liquophobic, preferably are hydrophobic and are also
significantly more porous than the reaction layer. Such material
may be present in sheet or fiber form, either woven or unwoven.
Suitable materials include polyamides, linear polyesters, such as
esters of ethylene glycol and terephthalic acid, polyolefins, such
as polypropylene, polyethylene, polymethylpentene, and
polyisobutylene, as well as copolymers formed by copolymerizing the
monomers used to form the aforementioned homopolymers, such as
ethylene-propylene copolymers. Mixtures or blends of such polymers
can also be used. The polyolefins are preferred and polypropylene
is particularly preferred.
The third or downstream layer constitutes a barrier layer which,
preferably, is in intimate contact with or, most preferably, is
adhered, bonded, or otherwise secured to the sealing layer,
particularly at the periphery of each well. It is this layer,
provided with at least one, and preferably only one, aperture
located substantially at the center of the well, which permits a
drop of liquid to pass from the well, under application of positive
or negative gauge pressure, i.e., super atmospheric pressure
applied upstream of the composite membrane or reduced pressure
applied downstream of the membrane, and to drop from the plate
without radial migration to another well. The apertures can also
serve as restriction means, controlling the flow rate of liquid
through the composite membrane. Thus, the barrier layer, because of
the nature of the material used therein, i.e., a highly
liquophobic, preferably hydrophobic, material, enhances isolation
between wells and forces the liquid passing through the composite
membrane to form small drops at the hole in the barrier layer.
These drops, rather than spreading radially when passing through a
porous or microporous liquophilic material (such as the material of
the reaction layer used alone or in conjunction with the sealing
layer) and forming a small contact angle with the surface of such
material, tend, when passing through the barrier layer, to form a
large contact angle with the surface of liquophobic material. Thus,
even if the device is raised at one end so that the bottom surface
is not completely horizontal, liquid drops passing through each
well tend to drop from the device rather than rolling to one side
and potentially contaminating fluid in an adjacent well.
The size and shape of the apertures formed in the barrier layer
depend on a number of variables including the porosity of the
barrier layer, the number of apertures in the barrier layer, the
porosity and number of apertures formed in the other layers
(discussed below), the number of wells, the flow rate sought, ease
of manufacturing, etc. However, when a single aperture is used in
the barrier layer of each well, it may be formed in a variety of
shapes. A suitable size for the aperture is about 1/32 to about 1/4
inch. An aperture or hole simply formed in the shape of an "X" in
the barrier layer, i.e., without a corresponding aperture in the
sealing layer or reaction layer, provides an operable system.
The material used to form the barrier layer should have a greater
liquophobicity than either the reaction layer or the sealing layer
and may be either microporous or nonporous. Another way of
expressing the suitability of a material as the barrier layer
relates to the wetting resistance characteristics of the material.
A suitable material should be capable of resisting a liquid
intrusion pressure greater than to the height of the column of
liquid above the barrier layer, i.e., the height of the liquid
placed in the well. Suitable materials include polyolefins, such as
polypropylene, polyhalogenated polyolefins, particularly
perfluorinated polyolefins, such as polytetrafluoroethylene, and
polyvinylidene difluoride, as well as sulfones.
Polytetrafluoroethylene is most preferred.
In addition to the aperture formed in the liquophobic barrier
layer, the reaction layer may also be provided with at least one,
and preferably no more than one, aperture. When a single aperture
is provided in the reaction layer, the diameter of the aperture may
be in the range of about 3 to about 100 microns. When present as a
plurality of apertures, each aperture may have a diameter of about
3 to about 100 microns. The choice of using a membrane either with
or without an aperture in the reaction layer is determined by such
factors as the source and type of vacuum or pressure apparatus
employed to force a liquid through the composite membrane, the
number of wells present in the device, the overall porosity of the
composite membrane, the porosity or bubble point characteristics of
the reaction layer, and the dimensions of the aperture(s) in the
barrier layer. All of these factors affect the rate of fluid flow
through the membrane at a given pressure drop.
The provision of one or more apertures in the reaction layer of the
composite membrane in each well assures total voiding of liquid
through the composite membrane when positive or negative gauge
pressures are employed. When the reaction layer is provided with an
aperture, a continuous, rather than an intermittent, application of
positive or reduced pressure is necessary since the vacuum or
positive pressure will be continuously bleeding away. However,
depending on the factors listed above, when a reaction layer having
no apertures passing therethrough is provided in the composite
membrane, a continuous application of positive or reduced pressure,
preferably the latter or a single or intermittent application of
positive or negative gauge pressures using, for instance, a syringe
and check valve combination may be employed. These latter
alternatives are generally preferred.
In some instances when manufacturing the composite membrane of the
present invention, it is desirable to initially provide each of the
layers with the appropriate number of apertures. Thus, a needle(s)
may be used to simultaneously form the apertures in each layer in
alignment with each other. However, with the porous sealing layer,
the aperture will usually not be much larger than the existing
pores and when a fibrous material is used, the fibers will
generally move back to the position they occupied before insertion
of the needle. In this embodiment, the diameters of the apertures
in the reaction and barrier layers are the same. This embodiment
provides for ease of manufacturing in that the apertures may be
formed after the composite membrane is assembled and the layers are
secured to one another.
Typically, reduced pressure or vacuum-assisted fluid flow is
accomplished with the present invention by means of a vacuum
manifold which is provided with a means for connection to a vacuum
source, such as a projecting tube which can be inserted into vacuum
tubing. Such manifolds also include a means to form an air-tight
seal between the well(s)-containing diagnostic plate device and the
manifold, such as a pliable gasket or the like. Such manifolds
commonly include a vertical wall portion, the internal surface of
which configurationally conforms to the outer surface of a vertical
wall portion of the plate device such that mating of the two wall
surfaces occurs. Other designs, however, may be employed, such as
the diagnostic plate device being provided with an internal wall
surface which mates with an external wall surface of the manifold.
Alternative structures in which mating surfaces of the manifold and
diagnostic plate device are provided and in which an air-tight seal
may be established may also be employed with the present
invention.
In use, a first test or reagent liquid is normally placed in each
of the wells of the diagnostic plate device, and the device is then
inserted into the vacuum manifold such that the gasket or sealing
means contacts mating surfaces in both the diagnostic plate device
and the manifold. Communication between a source of vacuum and the
manifold is then established. As the pressure in the manifold
decreases, the diagnostic plate device is drawn against the gasket,
improving the seal. As this occurs, liquid is drawn through the
wells into the vacuum line and to a waste trap. Solutions of
samples to be tested or reagents are then introduced into each of
the wells.
To provide a means for isolating and retaining fluids passing
through the diagnostic plate device of the present invention for
those situations in which it is desirable to conduct further tests
on the liquid, a second modified form of the diagnostic plate
device may be used within the vacuum manifold. The modified form of
the plate is positioned intermediate the vacuum manifold and the
diagnostic plate device in which test samples are analyzed. The
modified form of the device is one in which the wells have solid
impermeable bottoms, that is, the wells do not have open bottoms
covered with the composite membrane of the present invention but
rather the membrane is replaced with the same material from which
the walls of the wells are made or some similar material. In most
cases, the modified plate is of unitary construction in which the
side walls and bottom are formed integrally in a single step. The
sealing means is still maintained between the diagnostic plate
device of the present invention and the vacuum manifold when the
device is placed into operation.
In some situations, it may be desirable to use the diagnostic plate
device of the present invention in situations where a vacuum line
connected to a centralized source of vacuum or a vacuum pump is not
available. An embodiment of the present invention, nevertheless,
permits analysis in such instances. The manifold employed includes
a modified outlet means for connecting to a vacuum source.
Specifically, the outlet includes a one-way valve of a type
generally known to the valve art which permits fluids to be
withdrawn from the manifold but does not permit fluids to enter the
manifold. In addition, the connection means permits a syringe tip
to be inserted into the manifold so that a large syringe may be
used as the vacuum source. Such a means for connection to a syringe
tip may be a Luer locking device or the like. When a syringe is
used as the vacuum source, this will normally be able to displace a
much smaller volume of air than will a continuously operating
vacuum pump. Accordingly, the vacuum manifold and diagnostic plate
device adapted to be used with the manifold will be of a smaller
size with fewer than 96 wells which is a typical number for such
multiple well diagnostic plate devices.
As with the larger diagnostic plate device described above, the
periphery of the plate may have any suitable configuration as long
as adequate mating and sealing surfaces are provided between the
manifold and the diagnostic plate device. To sufficiently reduce
the volume of the manifold when the device is to be used with a
syringe as the source of vacuum, the periphery of the plate is
preferred to have a circular configuration, such as that shown in
FIG. 1, the wells also preferably being arranged in a circular
configuration. With such a configuration, various means may be
provided for the attachment of the plate to the manifold including
that described above in which the well-containing plate is merely
pressed into the manifold with a sealing means separating two
mating surfaces. Alternatively, in addition to sealing means,
threading or bayonet mounts may be provided on corresponding mating
surfaces of the plate and manifold. However, a preferred structure
is one that is disposable in which the diagnostic plate is formed
integrally with the manifold. Preferably, the integrally formed
structure also includes a means for trapping liquids in the
manifold. With this structure, possibly contaminated fluids are
trapped within the device, and the entire article, including
trapped liquids, could be disposed of while minimizing risks
associated with contamination.
FIGS. 1 to 3 illustrate preferred embodiments of the present
invention. As shown in the FIGS. 1 and 2, the device is formed from
a multi-well diagnostic plate or plate portion 1 and a vacuum
manifold or manifold portion 3. In one of the preferred embodiments
of the present invention, the elements 1 and 3 are formed as a
separate multiple well diagnostic plate and vacuum manifold, one
placed in the other and having a sealing surface formed
therebetween, as described above. Most preferred, however, is a
structure in which the device is formed integrally. In this latter
embodiment, no sealing means is necessary since what constitutes
the multiple well diagnostic plate portion 1 is not detachable from
the manifold portion 3. This embodiment is intended to be
disposable. Other than this feature and the vacuum connecting
means, the embodiments are substantially similar in most respects.
Thus, the diagnostic plate portion 1 is provided with an upper or
top wall 5 having located therein one or more wells 7. The wells
are defined by a wall portion 9 which is preferably vertical or
substantially vertical, although the wall portion may have a
downward taper. The well is provided with openings at both the top
and bottom ends. Sealed to the bottom end 19 of the well is
composite membrane 11 formed from an upper reaction layer 13, a
bottom barrier layer 15 having an aperture 29 passing therethrough,
and a sealing layer 17 located intermediate the reaction layer 13
and the barrier layer 15. As a result of the heat process used, a
dense, somewhat compacted, liquophobic zone 21 is provided at the
periphery of each well which prevents lateral flow or "cross-talk"
of fluids, particularly liquids, between wells.
The manifold portion 3 comprises a side wall or walls 23 and bottom
wall 25. The upper plate or wall 5, side wall 23, and bottom wall
25 define a housing in which the wells are located. A vacuum
connection means 27 is provided to communicate the housing or
manifold portion with a source of vacuum (not shown).
One preferred purpose of the sealing layer 17 is to seal the
barrier layer 15 to the reaction layer 13. Another function served
by the sealing layer is to provide a liquophobic seal at the
periphery or rim of each well where the composite membrane is
secured to the periphery of the opening at the bottom of the well.
Such a seal is typically accomplished by means of a heat, and
preferably combined with pressure, treatment. Heat-sealing methods
using radiant heat or ultrasonic sealing techniques with apparatus,
such as heater blocks or welding horns, respectively, may be
employed. It is the liquophobic seal formed at the periphery of the
opening of each well by such treatment which prevents liquid
passing through the composite membrane from diffusing or migrating
radially and possibly contaminating another well.
The embodiment of the present invention illustrated in FIG. 3
permits liquid which has passed through a well of a diagnostic
plate according to the present invention, such as that described
above, to be isolated from a test sample and retained. In this
embodiment, the reference numerals common to FIG. 3, and FIGS. 1
and 2, describe substantially the same elements. The major
distinction to be drawn between the embodiment of the present
invention illustrated in FIGS. 1 and 2 and that of FIG. 3 is the
inclusion within the manifold portion 3 of a second or receiving
plate 31 having a plate portion 35 with one or more wells 37
located therein, each well defined by a wall portion 39. The
dimensions and configurations of the plate portion 31, wall 35,
wells 37, and wall portion 39 are preferably the same or similar to
those of the corresponding top wall 5, wells 7, and wall portions 9
of the diagnostic plate portion 1. The major difference between the
upper diagnostic plate portion 1 and the second plate or receptacle
31 is that the latter substitutes a solid impermeable bottom 41 for
the composite membrane 11 of the former. The receptacle plate 31 is
arranged within the manifold 3 in such a manner that the second
plate (and particularly the well(s) 37 located therein) is aligned
with and in liquid receiving relationship to the first plate (and
particularly the wells 7 located in the diagnostic plate 1). This
permits liquid passing through each one of the wells 7 in the
diagnostic plate to be isolated from an adjacent well and, after
passing through the composite membrane 11, to be retained in a
solid bottom well 41 of the second plate 31 corresponding to the
well in the upper plate through which it just passed.
The composite membrane of the present invention may be assembled by
a variety of techniques. Thus, each of the separate layers may be
overlaid and the separate layers sealed to one another and to the
bottom of each well in a single heat-sealing procedure. This may be
accomplished by superposing the individual layers, i.e., the
reaction layer, the sealing layer, and the barrier layer, over the
open bottom ends of the wells such that the apertures in the
barrier layer and, where appropriate, other layers are located
substantially at the centers of each well. Heat is then applied
from the barrier layer side of the membrane to secure the separate
layers to one another and also to form a hydrophobic seal between
the composite membrane and the bottom edge surface of the wall of
each well. Heat is applied either directly in the form of radiant
heat by a heater block or as ultrasonic energy using a welding
horn. In this manner, the reaction layer is sealed to the edges of
each of the wells and, simultaneously, the sealing layer fuses the
reaction layer to the barrier layer.
To facilitate handling of the layers of material forming the
composite membrane, rather than superpose each layer individually
over the bottoms of the wells, it is preferred that the edges of
the sheets of material forming the layers may be welded or tacked
to one another to provide a preliminary composite membrane. The
sealing of the composite structure to the bottom edge surfaces of
the wells and possibly of the layers to each other occurring, as
indicated above, when heat is applied to the layers of material
superposed on the plate.
Alternatively, although less preferred, rather than adhering the
edges of the separate sheets such as by tacking or the like, the
three layers may be secured to one another to form the composite
membrane in a first step and the formation of a liquid-tight seal
to the bottom of the wells may be achieved in a second step. Other
alternatives include bonding the sealing layer to the reaction
layer in a first step, such as that described in U.S. patent
application Ser. No. 107,918, filed Oct. 13, 1987, which in turn is
a continuation of U.S. patent application Ser. No. 685,042, filed
Dec. 21, 1984, and incorporated herein by reference. According to
this method, microfibers are directed in a stream toward a sheet of
the reaction layer, generally after the fibers are extruded from a
fiberizing die and attenuated in one or more gas streams. The
microfibers are extruded at an elevated temperature and after
contacting the microporous reaction layer are cooled to form a
composite, two layer membrane containing a web of microfibers as a
sealing layer secured to the reaction layer. A similar procedure
may be employed in which the sealing layer is formed as a sheet
and, while at an elevated temperature, is placed in contact with a
sheet of the microporous reaction layer. The hydrophobic barrier
layer is then applied to the two layer composite membrane by a heat
bonding procedure.
In use, a solution containing the sample to be analyzed and one or
more reagent solutions may be added simultaneously or, in most
instances, preferably, sequentially to each well of the multiple
well diagnostic plate. Under appropriate temperature conditions,
the solutions are added to and removed from each well, in timed
sequence to allow sufficient time for reactions to take place in
solution or on the surface of the reaction layer before the
addition or removal, as appropriate, of a solution. In many
instances, molecular species, particularly macromolecular species,
such as nucleic acids, antibodies, antigens, or enzyme-labelled
conjugates are adsorbed. Weakly or non-adsorbed molecules are
removed from the surface of the composite membrane by washing
between steps with suitable buffers. When the reaction sequence is
completed, a labelled product, such as a visible colored or
fluorescent product, remains or is formed on the surface of the
membrane within the well.
To perform quantitative assays, the well(s)-containing plates are
placed in a suitable reading device, typically containing a source
and a detector of radiation. The colored or light emitting product
may then be quantitatively measured using either transmitted or
reflected light.
Solutions may be introduced to the well using a pipette. However,
after a suitable period for reaction to occur, the solutions may be
removed by application of vacuum or elevated pressure. Furthermore,
in some instances, it may be unnecessary to remove one solution
completely before the addition of another solution. Rather, one
solution may be added as the previous solution is being removed or,
in some instances, a second solution may be added while the first
is still present and the two solutions allowed to mix before
removal.
* * * * *